Study On The Heterologous Expression And Binding Activity Analysis Of Oxysterol-binding Protein In Aspergillus Oryzae And Its Mechanism Involved In Cholesterol Metabolism

Aspergillus oryzae(A.oryzae)3.042 is a kind of filamentous fungus microorganism widely used in fermented food,which is considered as a safe food grade fermentation strain.There are five Osh family proteins in A.oryzae 3.042,such as Osh1,Osh3,Osh5,Osh7 and ORP8.A large number of studies have shown that Osh proteins are very important to the transport of intracellular PIPs,cholesterol,oxysterol and other lipids.Therefore,it is of great significance to explore the structure and function of Osh proteins in A.oryzae 3.042 and its application for food fermentation.In this study,the function and structure of four Osh proteins(Osh3,Osh5,Osh7,ORP8)in A.oryzae 3.042 was analyzed,and the important mechanism of Osh proteins in the transport of cholesterol,ergosterol,ergosterol peroxide were researched,which lays a solid foundation for the follow-up research.The main research contents include:1?Bioinformatics analysis of Osh protein genes: The gene sequences of four Osh proteins in A.oryzae were obtained from the NCBI database,and six His tags were added to the N-terminal of the protein and then connected to p ET-28 vector.Four Osh genes were synthesized by Nanjing kingsley biotechnology co.,ltd..The structure and amino acid sequences of Osh proteins was analyzed by smart online software.The results showed that Osh3,Osh5,Osh7,ORP8 were short chain proteins with only ORD domain,and the conserved amino acid sequence of Osh proteins in Osh3 was“EKVSHRPV”,the conserved amino acid sequence in Osh5 was “EQVSHHPP”,the conserved amino acid sequence in Osh7 and ORP8 were “EQTSHHPP”.The molecular weight of four Osh proteins was analyzed by the protein analysis system protparam which named “ https://web.expasy.org/protparam/ ”.The results showed that Osh3 contains 485 amino acids and its molecular size was 53.47075 k D,Osh5 contains 417 amino acids and its molecular size was 46.26373 k D,Osh7 contains 445 amino acids and its molecular size was 50.09276 k D,ORP8 contains 431 amino acids and its molecular size was 47.79496 k D.2 ? Expression,purification and functional identification of recombinant Osh proteins: Because of the differences in the expression of four Osh proteins,Osh3 and Osh5 proteins can only be expressed in E.coli Transseta DE3 cells,while Osh7 and ORP8 proteins can be expressed in E.coli Transseta DE3 cells and E.coli BL21 DE3 cells.Therefore,the constructed prokaryotic expression vectors were transformed intoE.coli Transseta DE3 cells for protein expression.Four proteins were purified by His Ni-Agarose Resin.The results showed that only Osh3 was expressed at a higher level,and the amount of purification protein was larger,while the amount of other three Osh proteins was less.The purified Osh3 was quantified and its binding activity to 9 kinds of sterols was measured by MST.The results showed that Osh3 can be bound to these9 kinds of sterols,and the binding affinity of Osh3 to stigmasterol was strongest,while the binding affinity of Osh3 to 7-ketocholesterol was lowest.3?Functional analysis of Osh proteins involved in cholesterol metabolism and sterol transport: Through the relevant experiments of fermentation of media containing different carbon sources by A.oryzae,the contents of cholesterol,ergosterol and ergosterol peroxide in mycelium at different times were determined by HPLC.The results showed that the cholesterol content in the mycelium reached 43%of the total mycelium weight at 48 h,indicating that A.oryzae could efficiently utilize cholesterol.Further analysis showed that A.oryzae could promote the conversion of ergosterol into ergosterol peroxide by utilizing cholesterol.The content of ergosterol in the mycelium of cholesterol medium was significantly lower than that in the mycelium of glucose medium,while the content of ergosterol peroxides in the mycelium of cholesterol medium was significantly higher than that in the mycelium of glucose medium.Therefore,this study first showed that A.oryzae can utilize cholesterol,which can promote the conversion of ergosterol into ergosterol peroxide,and Osh proteins play an important role in this process.